Lipopolysaccharide Induces Upregulation of Neutral Endopeptidase 24.11 on Human Neutrophils: Involvement of the CD14 Receptor

1. As lipopolysaccharide is a major stimulator of neutrophil responses during Gram-negative bacterial infections, we studied its effect on the membrane expression of neutral endopeptidase 24.11/CD10 on neutrophils in a model of endotoxaemia in vitro. Lipopolysaccharide added to human whole-blood induced a marked and sustained CD10/neutral endopeptidase upregulation that was already detectable at 0.1 ng/ml and was maximal at a lipopolysaccharide concentration of 10 ng/ml. 2. We observed that neither tumour necrosis factor-α nor any newly synthesized protein was involved in the upregulation observed after 1 h incubation with 10 ng/ml lipopolysaccharide. 3. We further studied whether the lipopolysaccharide-induced CD10/neutral endopeptidase upregulation was mediated by lipopolysaccharide binding to the neutrophil CD14 receptor. Incubation of whole blood with an anti-CD14 monoclonal antibody before the addition of 0.1 ng/ml or 0.5 ng/ml lipopolysaccharide resulted in complete inhibition of CD10/neutral endopeptidase upregulation. In contrast, at a lipopolysaccharide concentration of 10 ng/ml, the anti-CD14 monoclonal antibody had an incomplete blocking effect. 4. The differential requirement for the CD14 receptor, depending on the lipopolysaccharide dose, was confirmed by the study of a patient suffering from paroxysmal nocturnal haemoglobinuria (in whom a complete defect in neutrophil CD14 expression was previously documented). 5. We finally confirmed these results using purified neutrophils, demonstrating that lipopolysaccharide-induced CD10/neutral endopeptidase upregulation depends on direct interaction with neutrophil CD14.

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Monoclonal antibody AHN-1 inhibits phagocytosis by human neutrophils

Blood ◽

10.1182/blood.v65.2.333.bloodjournal652333 ◽

1985 ◽

Vol 65 (2) ◽

pp. 333-339

Author(s):

KM Skubitz ◽

DJ Weisdorf ◽

PK Peterson

Keyword(s):

Monoclonal Antibody ◽

Lysosomal Enzyme ◽

Surface Proteins ◽

Superoxide Production ◽

Human Neutrophils ◽

Enzyme Release ◽

Specific Monoclonal Antibody ◽

Normal Human ◽

Major Surface

The granulocyte-specific monoclonal antibody, AHN-1, immunoprecipitates two major surface-iodinated proteins of 105,000 and 145,000 to 150,000 daltons from normal human neutrophils. In this study, the effect of AHN- 1 on a number of neutrophil functions was evaluated in vitro. Both complement- and antibody-mediated phagocytosis were inhibited when human neutrophils were pretreated with AHN-1 and opsonized bacteria were used as targets. The inhibition of phagocytosis was specific, in that lysosomal enzyme release and chemotaxis were not altered by treatment with AHN-1. AHN-1 did inhibit superoxide production by neutrophils in response to particulate stimuli, but not in response to the soluble stimulus, 12-O-tetradecanoylphorbol-13-acetate. The data indicate that one or both of these surface proteins may be important in the process of phagocytosis. AHN-1 should be useful in isolating and further characterizing the nature of these molecules.

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Effect of defensin HNP-1 of human neutrophils on production of tumor necrosis factor α by human blood monocytes in vitro

Bulletin of Experimental Biology and Medicine ◽

10.1007/bf00783767 ◽

1992 ◽

Vol 113 (5) ◽

pp. 709-712

Author(s):

N. I. Misuno ◽

T. S. Kolesnikova ◽

R. I. Lehrer ◽

T. Ganz ◽

N. N. Voitenok

Keyword(s):

Tumor Necrosis Factor ◽

Human Blood ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Human Neutrophils ◽

Blood Monocytes ◽

Factor Α ◽

Necrosis Factor

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Correlation between the stimulation of human neutrophil function by monoclonal antibody and by colony-stimulating factor

Blood ◽

10.1182/blood.v66.3.738.738 ◽

1985 ◽

Vol 66 (3) ◽

pp. 738-741 ◽

Cited By ~ 4

Author(s):

MA Vadas ◽

C Clarke ◽

NA Nicola ◽

AF Lopez

Keyword(s):

Monoclonal Antibody ◽

Neutrophil Function ◽

Common Mechanism ◽

Human Neutrophils ◽

Target Cells ◽

Colony Stimulating Factor ◽

Positive Correlation ◽

Stimulating Factor ◽

Stimulation Of

Abstract Purified human neutrophils from 48 individuals were tested for their capacity to kill antibody-coated target cells in vitro in the absence or presence of stimulating agents. The agents used to stimulate cytotoxic capacity were the monoclonal antibody (MAb) WEM-G1, colony- stimulating factor (CSF-alpha), or mononuclear cell supernatant (MNC- SN). There existed an heterogeneity among the neutrophils of different individuals in the capacity to kill target cells both in the unstimulated (“resting”) or the stimulated state. A positive correlation was found between the ability of neutrophils to kill in the “resting” state and their capacity to be stimulated by MAb WEM-G1, CSF- alpha, or MNC-SN. Furthermore, a strong positive correlation in the ability of neutrophils to be stimulated by the MAb WEM-G1 and either CSF-alpha (r = .76) or MNC-SN (r = .68), as well as between CSF-alpha and MNC-SN (r = .79) was demonstrated. No correlation was seen, however, between stimulation of neutrophil function in vitro and total blood leukocyte counts, neutrophil counts, monocyte counts, or intensity of binding of MAb WEM-G1. The observation that neutrophils respond to a similar extent to different types of stimulators, -such as cytokines (CSF-alpha and MNC-SN) and MAb, suggests that these two factors may be operating through a common mechanism and the degree of stimulation may reflect an intrinsic responsiveness of neutrophils that differs among individuals. Our results also suggest a potential clinical use of WEM-G1 in measuring neutrophil functional capacity in vitro and predicting the capacity to respond to CSF-like cytokines.

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Anti-Progressive Effect of Neutral Endopeptidase 24.11 (NEP/CD10) on Cervical Carcinoma in vitro and in vivo

Oncology ◽

10.1159/000087476 ◽

2005 ◽

Vol 69 (1) ◽

pp. 52-62 ◽

Cited By ~ 28

Author(s):

Mikio Terauchi ◽

Hiroaki Kajiyama ◽

Kiyosumi Shibata ◽

Kazuhiko Ino ◽

Shigehiko Mizutani ◽

...

Keyword(s):

Cervical Carcinoma ◽

Neutral Endopeptidase ◽

Endopeptidase 24.11

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Anti-Inflammatory and Antibacterial Activity Constituents from the Stem of Cinnamomum validinerve

Molecules ◽

10.3390/molecules25153382 ◽

2020 ◽

Vol 25 (15) ◽

pp. 3382 ◽

Cited By ~ 1

Author(s):

Chi-Lung Yang ◽

Ho-Cheng Wu ◽

Tsong-Long Hwang ◽

Chu-Hung Lin ◽

Yin-Hua Cheng ◽

...

Keyword(s):

Bacterial Infections ◽

Immune Cell ◽

Intraperitoneal Administration ◽

In Vitro Study ◽

Human Neutrophils ◽

Infection Model ◽

Ic50 Values ◽

Anti Inflammatory

One new dibenzocycloheptene, validinol (1), and one butanolide firstly isolated from the natural source, validinolide (2), together with 17 known compounds were isolated from the stem of Cinnamomum validinerve. Among the isolates, lincomolide A (3), secosubamolide (7), and cinnamtannin B1 (19) exhibited potent inhibition on both superoxide anion generation (IC50 values of 2.98 ± 0.3 µM, 4.37 ± 0.38 µM, and 2.20 ± 0.3 µM, respectively) and elastase release (IC50 values of 3.96 ± 0.31 µM, 3.04 ± 0.23 µM, and 4.64 ± 0.71 µM, respectively) by human neutrophils. In addition, isophilippinolide A (6), secosubamolide (7), and cinnamtannin B1 (19) showed bacteriostatic effects against Propionibacterium acnes in in vitro study, with minimal inhibitory concentration (MIC) values at 16 μg/mL, 16 μg/mL, and 500 μg/mL, respectively. Further investigations using the in vivo ear P. acnes infection model showed that the intraperitoneal administration of the major component cinnamtannin B1 (19) reduced immune cell infiltration and pro-inflammatory cytokines TNF-α and IL-6 at the infection sites. The results demonstrated the potential of cinnamtannin B1 (19) for acne therapy. In summary, these results demonstrated the anti-inflammatory potentials of Formosan C. validinerve during bacterial infections.

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Human Neutrophils Activated by Interferon-γ and Tumour Necrosis Factor-α Kill Entamoeba histolytica Trophozoites In Vitro

Journal of Leukocyte Biology ◽

10.1002/jlb.46.3.270 ◽

1989 ◽

Vol 46 (3) ◽

pp. 270-274 ◽

Cited By ~ 69

Author(s):

Michel Denis ◽

Kris Chadee

Keyword(s):

Tumour Necrosis Factor ◽

Entamoeba Histolytica ◽

Tumour Necrosis ◽

Interferon Γ ◽

Human Neutrophils ◽

Tumour Necrosis Factor Α ◽

Factor Α ◽

Necrosis Factor

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Monoclonal antibody-defined functional epitopes on the adhesion- promoting glycoprotein complex (CDw18) of human neutrophils

Blood ◽

10.1182/blood.v67.4.1007.1007 ◽

1986 ◽

Vol 67 (4) ◽

pp. 1007-1013 ◽

Cited By ~ 98

Author(s):

WJ Wallis ◽

DD Hickstein ◽

BR Schwartz ◽

CH June ◽

HD Ochs ◽

...

Keyword(s):

Monoclonal Antibody ◽

Active Site ◽

Bacterial Infections ◽

Quaternary Structure ◽

Alpha Subunit ◽

Human Neutrophils ◽

Glycoprotein Complex ◽

Dose Dependent Fashion ◽

Alpha Beta ◽

Dose Dependent

Abstract We have evaluated the functional and immunochemical activities of three monoclonal antibodies (MoAbs) minimally reactive with adherence- defective neutrophils (PMN) from a patient with recurrent bacterial infections. In studies with normal PMN, MoAbs OKM1 and 60.1 both precipitate the same 165kd alpha-subunit (alpha M) within an alpha-beta heterodimer complex (CD11). The CD11 complex is part of a larger complex composed of four glycoproteins (CDw18) precipitated by MoAb 60.3, with properties suggesting that the CDw18 complex is equivalent to the Mac-1, LFA-1, p150, 95 glycoprotein family implicated in adherence-dependent leukocyte functions. PMN adherence to endothelium, spreading on surfaces, aggregation, and phagocytosis of zymosan particles were all inhibited in a dose-dependent fashion by MoAb 60.1 (analogous to previous studies with MoAb 60.3) while MoAb OKM1 had no effect. These findings unify previously disparate observations and suggest that a functionally active site on the adherence promoting glycoprotein complexes CD11 and CDw18 is distant from the alpha M epitope recognized by MoAb OKM1 but closely associated with the alpha M epitope recognized by MoAb 60.1 and the beta-epitope (or epitope created by alpha-beta quaternary structure) recognized by MoAb 60.3.

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Ubc9 interacts with Lu/BCAM adhesion glycoproteins and regulates their stability at the membrane of polarized MDCK cells

Biochemical Journal ◽

10.1042/bj20060861 ◽

2007 ◽

Vol 402 (2) ◽

pp. 311-319 ◽

Cited By ~ 7

Author(s):

Emmanuel Collec ◽

Wassim El nemer ◽

Emilie Gauthier ◽

Pierre Gane ◽

Marie-Christine Lecomte ◽

...

Keyword(s):

Epithelial Cells ◽

Mdck Cells ◽

Ex Vivo ◽

Basolateral Membrane ◽

Physiological Role ◽

Direct Interaction ◽

Cytoplasmic Tail ◽

Protein Accumulation ◽

Membrane Expression

Lu (Lutheran) blood group and BCAM (basal cell adhesion molecule) antigens both reside on two gp (glycoprotein) isoforms, Lu and Lu(v13), that differ by the size of their cytoplasmic tail. They are receptors of laminin-10/11 and are expressed in RBCs (red blood cells), epithelial cells of multiple tissues and vascular endothelial cells. To gain more insights into the biological function of Lu/BCAM gps, we looked for potential partners of their cytoplasmic tail. We isolated Ubc9 (ubiquitin-conjugating enzyme 9) protein by screening a human kidney library using the yeast two-hybrid system. Lu/Ubc9 interaction was validated by GST (glutathione S-transferase) pull-down and co-immunoprecipitation experiments. Endogenous Ubc9 formed a complex with endogenous or recombinant Lu gp in A498 and MDCK (Madin–Darby canine kidney) epithelial cells respectively. Replacement of Lys585 by alanine in the Lu gp abolished in vitro and ex vivo interactions of Lu gp with Ubc9 protein. Lu K585A mutant transfected in MDCK cells exhibited a normal basolateral membrane expression but was overexpressed at the surface of polarized MDCK cells as compared with wild-type Lu. Pulse–chase experiments showed extended half-life of Lu K585A gp at the plasma membrane, suggesting an impaired endocytosis of this mutant leading to protein accumulation at the membrane. Furthermore, we showed that the ability of MDCK-Lu K585A cells to spread on immobilized laminin was dramatically decreased. Our results support a physiological role for the direct interaction between Lu gp and Ubc9 protein and reveal a role for this enzyme in regulating the stability of Lu gp at the cell membrane.

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CGS 34226, a thiol-based dual inhibitor of endothelin converting enzyme-1 and neutral endopeptidase 24.11

Clinical Science ◽

10.1042/cs103s098s ◽

2002 ◽

Vol 103 (s2002) ◽

pp. 98S-101S ◽

Cited By ~ 9

Author(s):

Y. Arco JENG ◽

Paula SAVAGE ◽

Michael E. BEIL ◽

Charles W. BRUSEO ◽

Denton HOYER ◽

...

Keyword(s):

Pressor Response ◽

Neutral Endopeptidase ◽

Intravenous Dose ◽

Renal Diseases ◽

Dual Inhibitor ◽

Endopeptidase 24.11 ◽

Anaesthetized Rats ◽

Ic50 Values

Endothelins (ETs) are potent vasoconstrictors and have been implicated in the pathogenesis of various cardiovascular and renal diseases. In contrast, atrial natriuretic peptide (ANP) is a potent vasorelaxant and diuretic agent, which is mainly degraded by neutral endopeptidase 24.11 (NEP) in vivo. Thus, compounds that can suppress the biosynthesis of ETs by inhibiting endothelin converting enzymes (ECEs), which catalyse the final step of post-translational processing of the vasoconstrictors, while simultaneously potentiating the levels of ANP by inhibiting NEP may have novel therapeutic utility. Through targeted screening of our compound library and subsequent optimization, CGS 34226 was identified as a potent, dual inhibitor of ECE-1 and NEP, inhibiting the enzymes with respective IC50 values of 11 and 4.6nM. In vivo, CGS 34226 suppressed the big endothelin-1 (big ET-1)-induced pressor response dose-dependently. At 15 and 90min after an intravenous dose of 30mg/kg in anaesthetized rats, this compound inhibited the big ET-1-induced effect by 79% and 65% respectively. In addition, CGS 34226 increased plasma ANP immunoreactivity by 120% up to 4h after an intravenous dose of 10mg/kg in conscious rats infused with ANP at a rate of 450ng/kg per min, intravenously. These results show that CGS 34226 is a potent dual inhibitor of ECE-1 and NEP in vitro and in vivo and that the compound may represent a novel agent for the treatment of cardiovascular and renal dysfunction.

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